The long-term objectives of this project are to understand the evolution of the T-box family of transcription factor genes, to determine their role in embryonic development., and to understand interrelationships between the genes in terms of the evolution of developmental mechanisms. In work completed during the previous funding period, we produced targeted mutations in five T-box genes, all of which have severe developmental consequences, and several which model human developmental disorders. In this proposal, we focus on the Tbx2 subfamily, Tbx2, Tbx3, Tbx4, and Tbx5 because of their expression in the allantois, a new structure in evolutionary terms, and their potential role in the evolution and development of paired appendages of tetrapods. We will use the mutant alleles we have already produced, new alleles, and combinations of alleles to study functional redundancy, unique function, and also whether the genes interact with one another. Our mutational analysis has already demonstrated dramatic affects on the development of the allantois and limbs and there is clear evidence of the importance of this gene sub- family to human health. Mutations in Tbx3 and Tbx5 cause two human developmental disorders, the ulnar-mammary, and Holt-Oram syndromes, respectively. We will use this group of genes to further our long-term objective of understanding interrelationships among T-box family members and how gene function evolved within this group. The Specific Aims will not only shed light on the genetic control of human development but also provide insight into the evolution of function within gene families. Specific Aim 1. Produce a multipurpose allele of Tbx5 to ablate gene function, allow real-time expression reporting and provide an allele that can be retargeted. Specific Aim 2. Produce a conditional allele of Tbx4 to study gene function late in development. Specific Aim 3. Investigate regulatory and genetic interactions between the genes of the Tbx subfamily, Tbx2, Tbx3, Tbx4, and Tbx5.